MRI image enhancement of bone and related tissue using complexes of paramagnetic cations and polyphosphonate ligands

ABSTRACT

Polyphosphonate ligands containing three or more phosphonate groups, combined with paramagnetic metal cations and administered in the form of pharmacologically acceptable salts, are useful as MRI contrast enhancement agents, which tend to localize in bone tissue without being conjugated to bone-specific biomolecules. Triazacyclononanes and tetraazacyclododecanes, with dihydroxyphosphorylmethyl or dihydroxyphosphorylethyl groups, optionally substituted at the methyl or ethyl bridges with alkyl, aryl, hydroxyl or amino groups, are particularly preferred.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.07/441,144, filed Nov. 27, 1989 now abandoned.

This invention lies in the field of magnetic resonance imaging, and isrelevant to the art of contrast enhancement agents used in connectionwith magnetic resonance imaging in medical diagnostics.

BACKGROUND OF THE INVENTION

The availability of magnetic resonance imaging (MRI) devices has led tothe use of MRI in medical examinations for the detection and diagnosisof disease states and other internal abnormalities. The continued useand development of MRI has stimulated interest in the development ofpharmaceutical agents capable of altering MRI images in diagnosticallyuseful ways. Pharmaceutical agents (MRI pharmaceuticals) which arecurrently favored by researchers in the field are suitably complexedparamagnetic metal cations. The use of pharmaceuticals in MRI imagingoffers major opportunities for improving the value of the diagnosticinformation which can be obtained.

Radiopharmaceuticals, which are used in radioisotopic imaging in amanner analogous to MRI pharmaceuticals, are a well developed field. Theknowledge existing in this field thus provides a starting point for thedevelopment of MRI pharmaceuticals. MRI pharmaceuticals must meetcertain characteristics, however, which are either not required or areconsiderably less critical in the case of radiopharmaceuticals. MRIpharmaceuticals must be used in greater quantities thanradiopharmaceuticals. As a result, they must not only produce detectablechanges in proton relaxation rates but they must also be (a)substantially less toxic, thereby permitting the use of greater amounts,(b) more water soluble to permit the administration of a higher dosagein physiologically acceptable volumes of solution, and (c) more stablein vivo than their radiopharmaceutical counterparts. In vivo stabilityis important in preventing the release of free paramagnetic metals andfree ligand in the body of the patient, and is likewise more criticaldue to the higher quantities used. For the same reasons, MRIpharmaceuticals which exhibit whole body clearance within relativelyshort time periods are particularly desirable.

Since radiopharmaceuticals are administered in very small dosages, therehas been little need to minimize the toxicity of these agents whilemaximizing water solubility, in vivo stability and whole body clearance.It is not surprising therefore that few of the ligands developed for useas components in radiopharmaceutical preparations are suitable for usein preparation of MRI pharmaceuticals. A notable exception is the wellknown ligand diethylene triamine pentaacetic acid (DTPA), which hasproved useful in forming complexes with both radiocations,pharmacologically suitable salts of which provided usefulradiopharmaceuticals, and paramagnetic cations such as gadolinium, whosepharmacologically suitable salts have proved useful as MRIpharmaceuticals.

Certain groups of radiopharmaceuticals tend to localize in bone tissue,and are thus of use in providing diagnostic information concerning bonedisorders. The properties of these agents which lead to theirlocalization in bone also allow for them to localize in soft tissuesbearing recognitions features in common with bone. Thus, manyradiopharmaceuticals which localize in bone are known, or believed, tolocalize in soft tissues which are found to have gross, microscopic orchemical evidence for deposition of calcium salts (e.g., metastaticcalcification), such as might occur in association with tissue injury.Thus, radiopharmaceuticals have shown localization in rhabdomyolysis ofvarious origins, in collagen disorders and in other injured tissues.Localization of such agents in areas of myocardial infarction is anexample of one application which has proven diagnostically useful.Radiopharmaceuticals which localize in bone have also been shown tolocalize in normal and malignant breast tissue, in pleural effusions, ininfarctions of the spleen and bowel, inflammatory bowel disease,radiation injury, metastatic calcification, and in a variety ofmalignant tumors, etc. Regardless of the mechanism of such localizationwe herein refer to the soft tissues which concentrate agents whichlocalize in bone as "bearing recognition features in common with bone."Exclusive of their localization in bone and tissues bearing recognitionfeatures in common with bone, these agents generally are distributed inthe extracellular fluid spaces of the body and therefore can be used toprovide information concerning the content and kinetics of theextracellular fluid of normal and abnormal tissues. One example of theclinical utility of this behavior is the detection of disruption of theblood brain barrier wherein extracellularly distributed agentsabnormally localize in the region of such disruption. Most of thepresently known agents which localize in bone are excreted from the bodyby the kidneys and therefore can be used to evaluate the renal excretorysystem. It is possible that such agents could be made more lipophilicsuch that they would be excreted by the liver, and therefore could beused to evaluate the hepatobiliary excretory system.

Agents which localize in bone and which provide MRI contrast enhancementcould be used to perform similar diagnostic procedures employingradiopharmaceuticals which localize in bone. Given the substantiallygreater spatial and temporal resolution of MRI techniques, as comparedto nuclear medical techniques, it is anticipated that useful diagnosticinformation could be obtained in abnormalities which were not detectedusing nuclear medical techniques, as for example in detection of smallareas of tissue damage and/or in small regions of deposition of calciumsalts. Moreover, fixation of MRI contrast enhancement agents in suchtissue would be expected to increase the relaxivity of the agent bydecreasing the molecular rotation rate thereby increasing signalintensity. However, known radiopharmaceutical agents which localize inbone are retained in the region of their deposition for very prolongedperiods of time making them unsuitable for use as MRI contrast agents.Moreover, these "bone seeking" pharmaceuticals which contain phosphonategroups are also known to be relatively strong chelators of calcium ionsand their administration at the dose and dose rate levels associatedwith the use of MRI contrast agents can be associated with induction ofacute hypocalcemia and attendant cardiac arrest. At present, MRIcontrast enhancement agents are not available which, while showingdiagnostically useful localization in bone, also show near quantitativewhole body clearance within acceptable time periods and which have lowtoxicity comparable to existing MRI contrast enhancement agents.

Most known MRI pharmaceuticals when administered in vivo do not bythemselves localize in specific tissues, but instead generallydistribute in extracellular fluid space in a nonspecific manner. Onemeans of achieving localization of these inherently nonspecificpharmaceuticals in selected tissues is by conjugation with biomoleculeswhich localize in the region of interest. Another means is byincorporating the complexes into bodies which localize in the region ofinterest. Hormones, albumins, liposomes, and antibodies have beenmentioned in such attachments or incorporation. See Gries, H., et al.,U.S. Pat. No. 4,647,447, Mar. 3, 1987.

SUMMARY OF THE INVENTION

It has now been discovered that preferential MRI image enhancement inbone tissue and other tissue bearing biospecific recognition features incommon with bone is achieved by the use of ligands which bear thisrecognition specificity, combined with paramagnetic metal cations andadministered in the form of pharmacologically acceptable salts. Thesecomplexes are fully suitable for use as MRI contrast enhancement agents,and tend to localize in bone tissue without either being conjugated tobone-specific biomolecules or being incorporated into bone localizingbodies. These agents show near quantitative whole body clearance and lowin vivo toxicity, and possess all of the requirements of MRI contrastenhancement agents. Ligands containing phosphonate groups are preferred,and further preferred are ligands containing three or more phosphonategroups, preferably bonded through alkyl bridges to nitrogen atoms.Cyclic groups are still further preferred, notably polyazacycloalkanes.Particularly preferred ligands are triazacyclononanes andtetraazacyclododecanes, with dihydroxyphosphorylmethyl ordihydroxyphosphorylethyl groups attached to the nitrogen atoms, thesegroups optionally substituted at the methyl or ethyl bridges with alkyl,aryl, hydroxyl or amino groups. Moreover, paramagnetic complexes ofphosphonate ligands derived from triazacyclanes represent a heretoforeunrecognized group of contrast agents fully suitable for use as generalMRI contrast enhancement agents.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

Among the ligands used in the practice of the present invention are theembodiments represented by the following formulas: ##STR1##

The R¹, R⁴, R¹¹, R²¹ and R³¹ groups in these formulas are phosphonategroups which may be the same or different on any particular species, andare generally represented by ##STR2## in which: R^(a), R^(b) and R^(c)are independently H, or alkyl or aryl groups which do not interfere withcomplexation; with complexation; and

n is zero or 1.

In this definition of R¹, R⁴, R¹¹, R²¹, and R³¹, certain classes ofcompounds are preferred. For those species in which n is 1, onepreferred class is that in which R^(a), R^(b) and R^(c) are each H; andR^(d) is H, OH, NH₂, C₁ -C₈ alkyl, phenyl or benzyl. Another preferredclass is that in which R^(a), R^(b) and R^(c) are each H; and R^(d) isH, OH, NH₂, C₁ -C₄ alkyl or benzyl. For those species in which n iszero, a preferred class is that in which R^(a) and R^(b) areindependently H, C₁ -C₄ alkyl or benzyl, while another preferred classis that in which R^(a) and R^(b) are independently H, C₁ -C₄ alkyl orbenzyl, and still another preferred class is that in which R^(a) is Hand R^(b) is H, C₁ - C₄ alkyl or benzyl.

The two R⁴ groups in Formula I may alternatively be joined together as asingle divalent group bridging the two end nitrogen atoms and having theformula ##STR3## in which R² and R³ are as defined below, and s is atleast 2, preferably 2 or 3.

The R², R¹², R²² and R³² groups in these formulas may also be the sameor different on any single species, and are each independently H oralkyl, aryl or mixed alkyl aryl groups (such as alkyl aryl ethers) whichdo not interfere with complexation.

Similarly, the R³, R¹³, R²³ and R³³ groups in these formulas may also bethe same or different on any single species, and are each independentlyH or alkyl, aryl or mixed alkyl aryl groups (such as alkyl aryl ethers)which do not interfere with complexation.

In Formula I, the subscripts p and q may be the same or different, andare each either 2 or 3. The subscript r is 0 to 3 inclusive, preferably0 to 2 inclusive, and most preferably 0 or 1.

In Formula II, t, u and v may be the same or different, and are eacheither 2 or 3. The value of w is at least 1, more preferably 1 to 4inclusive, still more preferably 1 to 3 inclusive, and most preferablyeither 1 or 2.

In preferred embodiments, all R¹, R¹¹, R²¹ or R³¹ groups on any singlespecies are the same. In further preferred embodiments, all R², R¹², R²²or R³² groups on any single species are the same, and all R³, R¹³, R²³or R³³ groups on any single species are the same. In still furtherpreferred embodiments, all R², R¹², R²² or R³² groups on any singlespecies are H, and all R³, R¹³, R²³ or R³³ groups on any single speciesare the same and are H or alkyl or aryl groups which do not interferewith complexation. In still further preferred embodiments, all R², R¹²,R²² or R³² groups as well as all R³, R¹³, R²³ or R³³ groups on anysingle species are H.

The complexation referred to in the descriptions of the alkyl and arylgroups is the complexation of the ligand with a paramagnetic metalcation to form a chelate. Alkyl and aryl groups which do not interferewith such complexation extend to a wide range in terms of size andconfiguration. Preferred alkyl groups are those having 1 to 8 carbonatoms, with 1 to 4 carbon atom alkyls more preferred, and methyl andethyl the most preferred. Both straight-chain and branched-chain alkylsare included. Preferred aryl groups are benzyl and phenyl, particularlybenzyl.

Paramagnetic metals of a wide range are suitable for complexation withthese ligands in the formation of the contrast enhancement agents of thepresent invention. These metals tend to focus in elements having atomicnumbers of 22-29 (inclusive), 42, 44 and 58-70 (inclusive), and haveoxidations states of 2 or 3. Of these, the ones having and atomic numberof 22-29 (inclusive) and 58-70 (inclusive) are preferred, and thosehaving atomic numbers of 24-29 (inclusive) and 64-68 (inclusive) aremore preferred. Examples of such metals are chromium (III), manganese(II), iron (II), iron (III), cobalt (II), nickel (II), copper (II),praseodymium (III), neodymium (III), samarium (III), gadolinium (III),terbium (III), dysprosium (III), holmium (III), erbium (III) andytterbium (III). Chromium (III), manganese (II), iron (III) andgadolinium (III) are particularly preferred, with iron (III) the mostpreferred.

Physiologically or pharmacologically compatible salts of the chelatesare formed by neutralizing acidic moieties on the chelate withphysiologically or pharmacologically compatible cations fromcorresponding inorganic and organic bases and amino acids. Examplesinclude alkali and alkaline earth metal cations, notably sodium. Furtherexamples are primary, secondary and tertiary amines, notably,ethanolamine, diethanolamine, morpholine, glucamine,N,N-dimethylglucamine, and N-methylglucamine (commonly referred to as"meglumine"). Examples of amino acid cations are lysines, arginines andornithines. As bases, these cations may be used in the form of oxides,hydroxides, carbonates, bicarbonates or any other base forms which willrelease the cations. Of the many embodiments of the present invention,one preferred class consists of the physiologically compatible saltswhich contain three equivalents of a physiologically compatible cationcombined with the trianionic complex of Fe(III) andN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane at a pHof 6.8 to 7.4. (The term "trianionic" in this context denotes an anionhaving a charge of -3.)

The compounds of the present invention are capable of preparation byknown procedures, some of which are described herein. The phosphonicacid, referred to herein as the "ligand," is first formed, followed bythe formation of the chelate complex and then the physiologicallycompatible salt.

According to a typical procedure, compounds with a methylene bridgebetween the N and P atoms (i.e., those in which n in the above formulaeis zero) are prepared by first treating the hydrobromide salt of theunsubstituted starting material (for example, 1,4,7-triazacyclononane or1,4,7,10-tetraazacyclododecane) with formaldehyde and diethyl phosphitein aqueous solution to form the perethyl phosphonate ester (i.e., allacid groups esterified with an ethyl group). The ester subsequently canbe hydrolyzed to the phosphonic acid ligand. Alkyl or aryl substitutionsare introduced on the methylene carbon by treatment of the perethylester with a strong base such as butyllithium at -78° C. and an alkyl oraryl halide.

Likewise, the preparation of compounds with an ethylene bridge betweenthe N and P atoms (n equaling 1) from the same unsubstituted startingmaterials is begun by treating the starting materials with diethyl2-bromoethylphosphonate in the presence of excess K₂ CO₃. This will formthe phosphonic acid perethyl esters, which are then hydrolyzed in thesame manner as the methylene bridge compounds.

Ethylene bridge compounds with a hydroxy substitution at the carbonadjacent to the P atom (i.e., as R^(d)) are prepared by using diethylepoxyethylphosphonate in place of the diethyl 2-bromoethylphosphonate,and base is not used in the reaction. Those skilled in the art willrecognize that similar compounds containing an amino substitution in theposition of the hydroxy substitution can be prepared similarly usingdiethyl ethyleniminophosphonate.

It was discovered that the procedure for combining the ligand with aparamagnetic metal cation to form the chelate complex is critical whenseeking to obtain a stable, chromatographically distinct species. Inparticular, for most of the complexes studied it was discovered that astable distinct species was obtained by heating a solution of the ligandand a water soluble compound of the metal cation to a temperature of atleast about 50° C., preferably at least about 80° C., and morepreferably to reflux (100° C. in an aqueous system), at a pH in excessof 7.0. In preferred embodiments, separation and purification areincorporated into the process of elevation of the pH and heating. Thus,after initially adding the acid form of the ligand and the halide formof the paramagnetic cation and heating, the pH is slowly elevated byslow addition of base in an amount of equivalents equal to the charge ofthe metal cation. Thus, when the metal cation is Mn(II), two equivalentsof base are added, and when the cation is Fe(III), three equivalents areadded. The neutral form of the complex can then usually be crystallizedas a solid from the solvent. While heating, the crystallized solid canbe added to water and sufficient base to neutralize all remaining labileprotonated sites of the complex. Following formation of thechromatographically distinct complex, the neutral form of the complexcan then typically be recrystallized following reacidification. Theoptimum temperature and base addition rate will vary from one metal ionto the next, and is readily determined by routine experimentation. Incertain cases, (e.g., the Fe(III) complex ofN,N',N"-tris(dihydroxyphosphorylethyl)-1,4,7-triazacyclononane where thecomplex forms multiple species on acidification), crystallization of theneutral complex from acid medium was not performed, and the desired saltwas obtained directly from solution.

Use of the procedure described typically results in species which arestable against degradation into multiple, chromatographically distinctspecies over time, and upon exposure to elevated temperature. The term"chromatographically distinct" is used herein to denote species which donot indicate separation into components when subjected to suitablechromatography.

Any water soluble form of the metal may be used. Notable examples arehalide salts. Chlorides are particularly preferred. Sparingly watersoluble oxides or salts may also be used. When oxides are used, additionof base is not needed to form the neutral form of the complex.

Physiological salts are prepared from the neutral forms of the complexesby conventional procedures. In a typical procedure, the desired salt ofthe complex is formed from the neutral form of the complex by additionof the required equivalent of the desired base. Heating until the pHstabilizes may be required. A solid form of the salt of the complex canbe obtained by conventional procedures, such as, for example,lyophilization, and the solid can be reconstituted withpharmacologically suitable aqueous solutions prior to administration topatients. The number of physiological cations present in the finalproduct is equal to the equivalents added during the step of baseaddition, and is readily confirmed by independent means such aselemental analysis or potentiometric titrations.

Administration of the MRI contrast agents of the present invention to apatient or subject on whom magnetic resonance imaging is to be performedis achieved by conventional procedures known in this art and disclosedin the literature. Aqueous solutions of the agents are most convenientlyused. The concentrations of the agents in these solutions and theamounts administered may vary widely, the optimum in each case varyingwith the strength of the magnetic moment of the paramagnetic metal inthe agent, the contrast enhancement strength of the chelate as a whole,the method of administration, the degree of contrast enhancement desiredor needed, and the age, weight and condition of the patient or subjectto whom administration is made. In most cases, best results are obtainedwith solutions at concentrations of about 0.05 to about 2.0 moles of theparamagnetic complex per liter, preferably about 0.1 to about 1.0 moleper liter. Likewise, best results in most cases are usually obtainedwith dosages ranging from about 0.01 to about 1.0 millimole of agent perkilogram of whole body weight (mM/kg), preferably from about 0.05 toabout 0.5 mM/kg. Administration may be achieved by any parenteral routeand method, most notably by intravenous administration. The rate ofadministration may likewise vary, best results generally being obtainedat rates ranging from about 0.1 mM/min/kg to about 0.1 mM/sec/kg.

The following examples are offered for purposes of illustration, and areintended neither to define nor limit the invention in any manner.

EXAMPLE 1 Syntheses of Dihydroxyphosphorylmethyl Species

This example illustrates the preparation of variousdihydroxyphosphorylmethyl compounds and complexes within the scope ofthe invention. Species based on both 1,4,7-triazacyclononane and1,4,7,10-tetraazacyclododecane are illustrated in parallel fashionstarting from the hydrobromide salts of 1,4,7-triazacyclononane and1,4,7,10-tetraazacyclododecane, respectively.

A. Synthesis of Perethyl Esters of Nitrogen-Substituted MethylenePhosphonates 1,4,7-Triazacyclononane and 1,4,7,10-Tetraazacyclododecane

The trihydrobromide salt of 1,4,7-triazacyclononane and the hydrobromidesalt of 1,4,7,10-tetraazacyclododecane were combined with 3.5equivalents, and 28 equivalents, respectively, of aqueous 37%formaldehyde solution. The mixtures were stirred for 15-30 minutes atroom temperature, after which time 3.5 equivalents and 14 equivalents,respectively, of diethyl phosphite were added to each solution, and thereaction mixtures were stirred at room temperature for an additional 2-5hours. Water was then added and the aqueous layers extracted five timeswith ethyl acetate. To the remaining water fractions, NaHCO₃ was addeduntil the solutions were of pH approximately 7.5. The solutions werethen continuously extracted with ether for 2-2.5 days. The products wereobtained as oils upon evaporation of the ether and as needed werepurified by chromatography, and were identified as the perethyl estersof 1,4,7-triazacyclononane and 1,4,7,10-tetraazacyclododecane,respectively, by NMR.

Those skilled in the art will recognize that this procedure can also beemployed to synthesize compounds derived from substituted forms of1,4,7-triazacyclononane and 1,4,7,10-tetraazacyclododecane where suchsubstitutions are on the ring carbons and consist of substituted orunsubstituted alkyl or aryl groups as listed above, retaining thesubstitutions in the corresponding positions on the product compounds.Those skilled in the art will further recognize that this procedure canbe employed in an analogous manner to synthesize other substituted andunsubstituted cyclical and linear polyamines.

B. Synthesis of Perethyl Esters of Nitrogen-Substituted MethylenePhosphonates Substituted with Benzyl Groups at the Methylene Carbon

In this procedure, one of the perethyl esters prepared in part A aboveis converted to an analog which contains a benzyl group attached to themethylene carbon.

The perethyl ester of 1,4,7-triazacyclononane prepared in part A above,dissolved in dry tetrahydrofuran, was combined with an excess ofbutyllithium at -78° C., and the reaction mixture was stirred for 30minutes at that temperature. An amount of benzyl bromide equal to thenumber of equivalents of butyllithium employed was then added withstirring. The mixture was then allowed to slowly warm to roomtemperature. After continued stirring at room temperature for anadditional 30 minutes, cold water was added and the aqueous layer wasextracted with diethyl ether. The ether was evaporated and the residuechromatographed on silica gel G60 70-230 mesh to obtain the perethylester ofN,N',N"-tris(dihydroxyphosphorylbenzylmethyl)-1,4,7-triazacyclononane,whose identity was established by proton NMR.

Those skilled in the art will recognize that this procedure can be usedto place other substituted or unsubstituted alkyl or aryl halides on themethylene carbon as well, using the appropriate alkyl or aryl halide.

C. Hydrolysis of the Perethyl Esters to the Phosphonic Acids

In this procedure, both perethyl esters of part A above were convertedto the corresponding phosphonic acids.

The perethyl esters were separately dissolved in concentratedhydrochloric acid and heated at 80° C. for six to eight hours. Theresulting solutions were evaporated to dryness, and the pure acid formswere obtained following crystallization from water or water/ethanol.Their identity as the acids was confirmed by proton NMR and elementalanalysis.

To further confirm the identity of the products, independent syntheseswere performed employing the method described by Polykarpov, Yu M., etal., "N,N',N"-Tris(phosphonomethyl)-1,4,7-triazacyclononane--a specificcomplexing agent for magnesium ion," Izv. Akad. Nauk SSSR. Ser. Khim.,1982, (7), 1669-70. The products obtained were found to be identical byNMR to those obtained by the synthesis described above.

EXAMPLE 2 Syntheses of Dihydroxyphosphorylethyl Species

This example illustrates the preparation of certaindihydroxyphosphorylethyl analogs of the compounds prepared in Example 1.Species based on both 1,4,7-triazacyclononane and1,4,7,10-tetraazacyclododecane are again illustrated in parallelfashion.

A. Synthesis of Per-N-Substituted Dihydroxyphosphorylethyl Phosphonates

Diethyl 2-bromoethylphosphonate, prepared by procedures described in theliterature, was reacted separately with the hydrobromide salts of1,4,7-triazacyclononane and 1,4,7,10-tetraazacyclododecane in water inthe presence of excess K₂ CO₃ at 80° C. for 4-5 hours. The water wasthen removed by evaporation and chloroform was added to the solids toremove the product from the inorganic salts. The products were purifiedby chromatography employing neutral alumina and an elution solvent of10% methanol in chloroform. The perethyl ester groups were removed byhydrolysis using HCl as described in part C of Example 1 above. The pureproducts were obtained by crystallization from 10% ethanol in water, andtheir identity was established asN,N',N"-tris(dihydroxyphosphorylethyl)-1,4,7-triazacyclononane andN,N',N",N'"-tetrakis(dihydroxyphosphorylethyl)-1,4,7,10-tetraazacyclododecane,respectively, by proton NMR and elemental analysis after accounting forwater of hydration.

Those skilled in the art will recognize that this procedure can beemployed in an analogous manner to synthesize similar compounds havingsubstituted or unsubstituted alkyl or aryl substitutions on the ethylenecarbon atoms by employing correspondingly substituted diethyl2-bromoethylphosphonate.

B. Synthesis ofN,N',N"-tris(dihydroxyphosphorylhydroxyethyl)-1,4,7-triazacyclononane

Diethyl epoxyethyl phosphonate, prepared employing known procedures, wascombined with a solution of 1,4,7-triazacyclononane in methanol at roomtemperature, using 3.3 equivalents of the diethyl epoxyethylphosphonate. The solution was stirred for six hours at 40°-50° C. Themethanol was evaporated and the residue was dissolved in water. Theexcess epoxide was then extracted with diethyl ether and the water layerwas evaporated. The residue was purified by chromatography using neutralalumina by first eluting the column with chloroform followed by 10%methanol in chloroform. The perethyl ester groups were removed byhydrolysis in HCl as described above. The pure product was obtained bycrystallization from 10% ethanol in water. The identity of the productwas established as that ofN,N',N"-tris(dihydroxyphosphorylhydroxyethyl)-1,4,7-triazacyclononane byproton NMR and elemental analysis after accounting for three moleculesof water of hydration.

Those skilled in the art will recognize that thetris-dihydroxyphosphorylaminoethyl analog is similarly prepared by thesame procedure, using diethyl ethylenimino phosphonate in place of thediethyl epoxyethyl phosphonate, and that similar compounds bearingsubstituted or unsubstituted alkyl or aryl substitutions on the ethylenecarbon atoms are prepared analogously by employing correspondinglysubstituted forms of diethyl epoxyethyl phosphonate or diethylethylenimino phosphonate. The same procedure can likewise be used tosynthesize hydroxyphosphoryl hydroxyethyl and hydroxyphosphorylaminoethyl derivatives of other polyamines.

EXAMPLE 3 Preparation of Metal Cation Complexes A. Fe(III) and Cr(III)Complexes ofN,N',N"-Tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane

In separate syntheses,N,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane in waterwas combined with an equivalent of a water soluble salt of theappropriate metal cation to be included in the complex (i.e., FeCl₃ •6H₂O or CrCl₃ •6H₂ O). The mixtures were heated in a reflux apparatus at100° C. while base was slowly added in an amount equal to "n"equivalents, where "n" equals the charge of the metal cation. TheFe(III) complex crystallized from the aqueous solution, permittingrecovery in high yield. The Cr(III) complex crystallized upon additionof ethanol. The crystallized complex in each case was added to freshwater and sufficient base was added to yield a final pH of>10.0. In thecase of the Fe(III) complex, the resulting solution was heated to 100°C. while in the case of the Cr(III) complex, the resulting solution washeated to 140° C. (under pressure). Heating was continued in each caseuntil a single chromatographic species was obtained.

The solutions were then cooled and filtered to remove solids, and acidwas added to the filtrate to crystallize or precipitate the complex asbefore. Additional crystallizations of the complex were performed fromwater or water/ethanol. The purity of each complex was established bythin layer chromatography (TLC). The identity of each product wasestablished by elemental analysis.

B. Fe(III) Complexes ofN,N',N"-Tris(dihydroxyphosphorylethyl)-1,4,7-triazacyclononane andN,N',N"-Tris(dihydroxyphosphorylhydroxyethyl)-1,4,7-triazacyclononane

These complexes were prepared following modifications of the procedureof part A above. When solutions of the Fe(III) complex ofN,N',N"-tris(dihydroxyphosphorylethyl)-1,4,7-triazacyclononane wereacidified, additional products were noted on chromatographic analysis.Consequently, the final recrystallization step requiring acidificationwas eliminated, and the neutral form of the complex was not isolated asa solid. The tri-sodium salt of the product was purified in an ionexchange column, and the inorganic salts were removed by use of an LH20column. In the case of the Fe(III) complex ofN,N',N"-tris(dihydroxyphosphorylhydroxyethyl)-1,4,7-triazacyclononane, asingle chromatographically distinct product was not obtained using thisprocedure.

C. Mn(II) and Mn(III) Complexes ofN,N',N"-Tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane

The procedure for these complexes was modified due to the ease withwhich redox reactions occurred. The tetra-sodium salt of the Mn(II)complex was formed directly by adding six equivalents of NaOH to a 1:1mixture of ligand and MnCl₂ in water, followed by crystallization of thesalt of the complex by addition of ethanol and cooling. To prepare thetri-sodium salt of the Mn(III) complex, a stoichiometric quantity ofpersulfate ion was added to the tetra-sodium salt of the Mn(II) complex,and the reaction allowed to stand at room temperature until all of theMn(II) had oxidized to Mn(III). The product was purified by passagethrough an ion exchange column, and inorganic salts were removed bypassage through an LH-20 column. Both products were characterized assingle, chromatographically distinct products on TLC.

D. Gd(III) Complexes ofN,N',N"-Tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane,N,N',N",N'"-Tetrakis(dihydroxyphosphorylmethyl)-1,4,7,10-tetraazacyclododecaneandN,N',N",N'"-Tetrakis(dihydroxyphosphorylethyl)-1,4,7,10-tetraazacyclododecane

The tri-sodium salt of the Gd(III) complex ofN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane was madedirectly from GdCl₃ •6H₂ O and the acid form of the ligand by addingequivalent amounts of each to water and heating at 100° C. When thesolution was clear, six equivalents of NaOH were added slowly, and thesolution was heated for an additional five days. After centrifugation toremove the small amount of residual solids, the solution was dried togive a solid which was characterized as a single, chromatographicallydistinct product on TLC.

The neutral form of the Gd(III) complex ofN,N',N",N'"-tetrakis(dihydroxyphosphorylmethyl)-1,4,7,10-tetraazacyclododecanewas made from GdCl₃ •6H₂ O and the acid form of the ligand by addingequivalent amounts of each to water, followed by slow addition of threeequivalents of NaOH. Heating at 90° C. resulted in formation of agelatinous precipitate. Heating was continued until no furtherprecipitate formed, and the reaction mixture was allowed to cool to roomtemperature. The precipitate which formed as a result was isolated bycentrifugation and washed with water. The washed precipitate was addedto water, the pH was raised above 11 by addition of NaOH, and theresulting clear solution was heated overnight at 100° C. The solutionwas acidified to pH<3.0 with concentrated HCl, and was concentrated andcooled, yielding solids which were separated by centrifugation.

The pentameglumine salt of the Gd(III) complex ofN,N',N",N'"-tetrakis(dihydroxyphosphorylethyl)-1,4,7,10-tetraazacyclododecanewas made directly from Gd₂ O₃ and the acid form of the ligand by adding0.5 molecular equivalents of the former and 1.0 molecular equivalents ofthe latter to water and heating at 90° C. until a clear solution wasobtained. After filtration, five equivalents of meglumine were added tothe filtrate, and the reaction was heated at 100° C. for 20 hours. Aftercooling, the reaction mixture was brought to dryness to obtain the solidproduct.

EXAMPLE 4 Preparation of Physiological Salts A. Sodium and MeglumineSalts of Fe(III) Complex withN,N',N"-Tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane

The solid form of the Fe(III) complex ofN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane, thecomplex whose preparation is described in Example 3, part A above, wasadded to water at room temperature. Sodium hydroxide or meglumine wereadded to separate solutions of the neutral Fe(III) complex ofN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane untilthe solutions maintained a pH of 7.0 to 7.4. The solutions were thenlyophilized to obtain the solid physiological salts of the complex.These solids, when reconstituted with a suitable aqueous solvent priorto use, are suitable for in vivo administration. In each case, for thesodium salts of the complexes, potentiometric titration demonstratedthat the principal form of the complexes at pH 7.0 to 7.4 was thetrianion of the complex, and thus that the principal salt forms at thispH were the trisodium and the trimeglumine salt.

B. Meglumine Salt of Cr(III) Complex withN,N',N"-Tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane

The solid form of the Cr(III) complex ofN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane, thecomplex whose preparation is described in Example 3, part A above, wastreated with three equivalents of meglumine in a fashion comparable tothat described in Example 4, Part A above. Potentiometric titration ofthe sodium salt of this complex demonstrated that the principal form ofthe resulting salt at pH 7.0 to 7.4 was the trianion.

Those skilled in the art will recognize that other salts of the subjectcomplexes can be obtained employing similar procedures.

EXAMPLE 5 Product Evaluation

In the following studies, the test species are referred to as follows:

                  TABLE 1                                                         ______________________________________                                        Test Species                                                                                          Para-    Physiologically                                                      magnetic Compatible                                   Ref. Ligand             Cation   Cation                                       ______________________________________                                        A    N,N',N"-tris(dihydroxy-                                                                          Fe(III)  trisodium                                         phosphorylmethyl)-1,4,7-                                                      triazacyclononane                                                        B    N,N',N"-tris(dihydroxy-                                                                          Fe(III)  tri-meglumine                                     phosphorylmethyl)-1,4,7-                                                      triazacyclononane                                                        C    N,N',N"-tris(dihydroxy-                                                                          Fe(III)  trisodium                                         phosphorylethyl)-1,4,7-                                                       triazacyclononane                                                        D    N,N',N"-tris(dihydroxy-                                                                          Cr(III)  tri-meglumine                                     phosphorylmethyl)-1,4,7-                                                      triazacyclononane                                                        E    N,N',N"-tris(dihydroxy-                                                                          Mn(II)   tetra-sodium                                      phosphorylmethyl)-1,4,7-                                                      triazacyclononane                                                        F    N,N',N"-tris(dihydroxy-                                                                          Mn(III)  trisodium                                         phosphorylmethyl)-1,4,7-                                                      triazacyclononane                                                        G    N,N',N"-tris(dihydroxy-                                                                          Gd(III)  trisodium                                         phosphorylmethyl)-1,4,7-                                                      triazacyclononane                                                        H    N,N',N",N'"-tetrakis                                                                             Gd(III)  penta-                                            (dihydroxyphosphorylethyl)- meglumine                                         1,4,7,10-tetraazacyclododecane                                           ______________________________________                                    

A. Water Solubility

All of the test species listed above were dissolved in water,demonstrating solubility at concentrations sufficient to be useful aspharmaceutical agents. In particular, test species A and B provedsoluble in water at concentrations exceeding 50% (weight/volume).

B. Stability

TLC was performed on test species A and D, both before and after heatingat 100° C. for two hours. A single, chromatographically distinct spotwhich did not vary as a result of the heating was observed in bothcases.

C. Toxicity

Physiological salts of the various ligand/metal cation complexesdescribed herein were administered intravenously to mice. The mice wereobserved for two weeks following such administration, and the resultsare listed in Table 2 below. In this data, the administered dose isexpressed as mM of complex per kg whole body weight (mM/kg), and theadministration rate is expressed as mM of complex administered persecond or per minute per kg whole body weight (mM/sec/kg or mM/min/kg).The mice were considered to have "survived" administration of each agentif they were alive at the end of the two-week period.

                  TABLE 2                                                         ______________________________________                                        Toxicity Test Results                                                              Test                                                                     Test Species Dose        Rate       Survived?                                 ______________________________________                                        (1)  A       11.8   mM/kg  0.4 mM/sec/kg                                                                              yes                                   (2)  B       9.8    mM/kg  0.7 mM/min/kg                                                                              yes                                   (3)  C       10.0   mM/kg  2.0 mM/min/kg                                                                              yes                                   (4)  D       2.9    mM/kg  0.5 mM/sec/kg                                                                              yes                                   (5)  E       2.9    mM/kg  1.1 mM/min/kg                                                                              yes                                   (6)  F       8.0    mM/kg  2.6 mM/min/kg                                                                              yes                                   (7)  G       3.1    mM/kg  0.8 mM/min/kg                                                                              yes                                   ______________________________________                                    

When complexes of Fe(III) withN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane (TestSpecies A) were prepared employing alternate complexation procedures andwhich contained multiple forms of the complex, as detected by TLC, thetoxicity observed was substantially greater than that observed withpreparations of this complex prepared employing the procedure describedherein and which showed a single, chromatographically distinct producton TLC.

D. In Vivo Distribution And Whole Body Clearance Studies

Radioisotopically labeled analogs of test species selected from thoselisted above were synthesized, using radioisotopes of Fe(III) (iron-59),Cr(III) (chromium-51) and Gd(III) (gadolinium-153), and employing thegeneral synthesis procedures described above. The resulting complexeswere subjected to radiochromatography to insure acceptable radiopurityand identity with the parent complexes. They were then administeredintravenously to mice in order to measure in vivo distribution and wholebody clearance.

The location of the test species in the mice's bodies and the rate atwhich the test species were cleared from the mice's bodies afteradministration were determined by radioassay of tissues and whole bodycounting, both performed by conventional gamma ray counting techniques.Concentration of activity in tissues was determined as activity per gramof tissue, and whole body counts were expressed as the percentage ofwhole body activity at a given time with respect to whole body activityimmediately after injection. The results were as follows:

1. Radioisotopes of Test Species A and B.

In vivo distribution. Within 2 minutes following administration,evidence of concentration of radioactivity in bone and kidneys was seen.By measurements taken one hour after administration, the ratio of theconcentration of the test species in bone to that in whole blood wasgenerally over 25:1, while the ratio of their concentration in kidney tothat in blood was generally over 10:1. Even after 24 hours, when lessthan 5% of the administered dose remained in the body (see below), ahigh ratio of concentration of the test species in bone and kidney tothat in blood was maintained. In mice who had a tibia broken two to fourweeks prior to the study, the tibia which had suffered the fractureshowed significantly greater accumulation of activity than that whichwas measured in the contralateral normal tibia. All mice showed very lowactivity in the brain at all times following their administration.

Whole Body Clearance. Within 24 hours of administration, over 95% ofboth test species had been cleared from the body, almost exclusivelythrough the urine.

2. Radioisotope of Test Species C.

In vivo distribution. One hour after administration, the measuredbone-to-blood concentration ratio was greater than 4.5:1, and themeasured kidney-to-blood ratio was greater than 5.5:1. A very lowconcentration of this agent in brain was noted within this first hour.

Whole Body Clearance. Within 24 hours of administration, over 95% of thetest species had been cleared from the body.

3. Radioisotope of Test Species D.

In vivo distribution. One hour after administration, the measuredbone-to-blood concentration ratio was greater than 6:1, and the measuredkidney-to-blood ratio was greater than 10:1. A very low concentration ofagent in brain was noted within this first hour.

Whole Body Clearance. Within 24 hours of administration, over 95% of thetest species had been cleared from the body, almost exclusively throughthe urine.

E. Relaxivity Measurements

Measurements of proton longitudinal relaxivity (1/τ₁) were performed onsome of the test species listed above, and compared with those obtainedusing the following complexes outside the scope of this invention: (i)Gd(III) with diethylenetriamine pentaacetic acid (DTPA), and (ii)Fe(III) with N,N'-ethylenebis[(2-hydroxyphenyl)-glycinate] (EHPG). Allmeasurements were obtained using a Bruckner PC/20 Minispec deviceoperating at 20 MHz. All samples were dissolved in 0.1M phosphate bufferat pH 7.2.

The proton longitudinal relaxivity of Test Species A was two to threetimes greater than that obtained for the Fe(III) complex of EHPG andbetween 40 and 45% of that obtained for the Gd(III) complex of DTPA.Similar results were obtained for Test Species C.

F. Relative Equilibrium Constant Measurements

The highly colored Fe(III) complex of EHPG (used for comparison in partE of this example) was dissolved in 0.25M phosphate buffer at pH 7.2,and an equimolar quantity ofN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane wasadded. The solution was heated overnight at 100° C. The resultingsolution was devoid of the purple Fe(III) EHPG complex color, and TLCshowed only the presence of the Fe(III)N,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane complex.

The reverse experiment was also performed. The Fe(III) complex ofN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane was thusdissolved in 0.25M phosphate buffer at pH 7.2, and an equimolar quantityof EHPG was added. The solution was heated overnight at 100° C. As inthe first experiment, the resulting solution was devoid of the purplecolor of Fe(III) EHPG, and TLC showed only the presence of the Fe(III)N,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane complex.

These results demonstrate the relative stability of the Fe(III)N,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane complexin comparison to the Fe(III) EHPG complex.

The foregoing is offered primarily for purposes of illustration. It willbe readily apparent to those skilled in the art that further variations,substitutions and modifications in the substances and proceduresinvolved in the invention beyond those specifically disclosed herein maybe made without departing from the spirit and scope of the invention.

What is claimed is:
 1. A method of preferentially enhancing magneticresonance image contrast, said method comprising administering to saidpatient an effective amount of a pharmaceutical agent comprising aphysiologically compatible salt of the complex produced by the additionof a suitable paramagnetic metal cation to a chelator having the formula##STR4## in which: the R¹ moieties are each independently ##STR5## inwhich R⁵, R⁶ and R⁷ are independently selected from the group consistingof H and alkyl and aryl groups which do not interfere with complexation;R⁸ is selected from the group consisting of H, OH, NH₂, and alkyl andaryl groups which do not interfere with complexation; and m is zero or1;the R² moieties are each independently selected from the groupconsisting of H and alkyl and aryl groups which do not interfere withcomplexation; the R³ moieties are each independently selected from thegroup consisting of H and alkyl and aryl groups which do not interferewith complexation; p is 2 or 3; q is 2 or 3; r is 0 or 1; and the R⁴moieties together form a single divalent group having the formula##STR6## in which R² and R³ are as defined above, and s is at least 2.2. A method in accordance with claim 1 in which s is 2 or
 3. 3. A methodof preferentially enhancing magnetic resonance image contrast, saidmethod comprising administering to said patient an effective amount of apharmaceutical agent comprising a physiologically compatible salt of thecomplex produced by the addition of a suitable paramagnetic metal cationto a chelator having the formula ##STR7## in which: the R¹¹ moieties areeach independently ##STR8## in which R¹⁴, R¹⁵ and R¹⁶ are independentlyselected from the group consisting of H and alkyl and aryl groups whichdo not interfere with complexation; R¹⁷ is selected from the groupconsisting of H, OH, NH₂, and alkyl and aryl groups which do notinterfere with complexation; and n is zero or 1;the R¹² moieties areeach independently selected from the group consisting of H and alkyl andaryl groups which do not interfere with complexation; the R¹³ moietiesare each independently selected from the group consisting of H and alkyland aryl groups which do not interfere with complexation; t is 2 or 3; uis 2 or 3; v is 2 or 3; and w is
 1. 4. A method in accordance with claim3 in which R¹⁴, R¹⁵ and R¹⁶ are independently selected from the groupconsisting of H, C₁ -C₈ alkyl, phenyl and benzyl; and R¹⁷ is selectedfrom the group consisting of H, OH, NH₂, C₁ -C₈ alkyl, phenyl andbenzyl.
 5. A method in accordance with claim 3 in which R¹⁴, R¹⁵ and R¹⁶are independently selected from the group consisting of H, C₁ -C₄ alkyland benzyl; and R¹⁷ is selected from the group consisting of H, OH, NH₂,C₁ -C₄ alkyl and benzyl.
 6. A method in accordance with claim 3 in whichR¹⁴, R¹⁵ and R¹⁶ are each H; R¹⁷ is selected from the group consistingof H, OH, NH₂, C₁ -C₈ alkyl, phenyl and benzyl; and n is
 1. 7. A methodin accordance with claim 3 in which R¹⁴, R¹⁵ and R¹⁶ are each H; R¹⁷ isselected from the group consisting of H, OH, NH₂, C₁ -C₄ alkyl andbenzyl; and n is
 1. 8. A method in accordance with claim 3 in which R¹⁴,R¹⁵, R¹⁶ and R¹⁷ are each H; and n is
 1. 9. A method in accordance withclaim 3 in which R¹⁴ and R¹⁵ are independently selected from the groupconsisting of H, C₁ -C₈ alkyl, phenyl and benzyl; and n is zero.
 10. Amethod in accordance with claim 3 in which R¹⁴ and R¹⁵ are independentlyselected from the group consisting of H, C₁ -C₄ alkyl and benzyl; and nis zero.
 11. A method in accordance with claim 3 in which R¹⁴ and R¹⁵are each H; and n is zero.
 12. A method in accordance with claim 3 inwhich the R¹² and R¹³ moieties are each independently selected from thegroup consisting of H, C₁ -C₈ alkyl, phenyl and benzyl.
 13. A method inaccordance with claim 3 in which the R¹² and R¹³ moieties are eachindependently selected from the group consisting of H, C₁ -C₄ alkyl andbenzyl.
 14. A method in accordance with claim 3 in which the R¹²moieties are each H; and the R¹³ moieties are each independentlyselected from the group consisting of H, C₁ -C₈ alkyl, phenyl andbenzyl.
 15. A method in accordance with claim 3 in which the R¹²moieties are each H; and the R¹³ moieties are each independentlyselected from the group consisting of H, C₁ -C₄ alkyl and benzyl.
 16. Amethod in accordance with claim 3 in which the R¹² moieties are each H;and the R¹³ moieties are each independently selected from the groupconsisting of H and C₁ -C₄ alkyl.
 17. A method in accordance with claim3 in which the R¹² moieties are each H; and the R¹³ moieties are eachindependently selected from the group consisting of H and methyl.
 18. Amethod in accordance with claim 3 in which the R¹² moieties are each H;and the R¹³ moieties are each H.
 19. A method in accordance with claim 3in which t, u and v are each
 2. 20. A method in accordance with claim 3in which said paramagnetic metal cation is a cation of an element havingan atomic number of 22 to 29 or 58 to
 70. 21. A method in accordancewith claim 3 in which said paramagnetic metal cation is a cation of anelement selected from the group consisting of chromium, manganese, ironand gadolinium.
 22. A method in accordance with claim 3 in which saidphysiological compatible salt is comprised of said complex incombination with at least one cation selected from the group consistingof sodium and N-methylglucamine.
 23. A method of preferentiallyenhancing magnetic resonance image contrast in bone tissue and othertissue of a patient bearing recognition features in common with bonetissue, said method comprising administering to said patient aneffective amount of a pharmaceutical agent comprising a physiologicallycompatible salt of the complex produced by the addition of a suitableparamagnetic metal cation to a chelator having the formula ##STR9## inwhich: the R²¹ moieties are each independently ##STR10## in which R²⁴,R²⁵ and R²⁶ are independently selected from the group consisting of Hand alkyl and aryl groups which do not interfere with complexation; R²⁷is selected from the group consisting of H, OH, NH₂, and alkyl and arylgroups which do not interfere with complexation; and x is zero or 1;theR²² moieties are each independently selected from the group consistingof H and alkyl and aryl groups which do not interfere with complexation;and the R²³ moieties are each independently selected from the groupconsisting of H and alkyl and aryl groups which do not interfere withcomplexation.
 24. A method in accordance with claim 23 in which R²⁴, R²⁵and R²⁶ are independently selected from the group consisting of H, C₁-C₄ alkyl and benzyl; and R²⁷ is selected from the group consisting ofH, OH, NH₂, C₁ -C₄ alkyl and benzyl.
 25. A method in accordance withclaim 23 in which R²⁴, R²⁵ and R²⁶ are each H; R²⁷ is selected from thegroup consisting of H, OH, NH₂, C₁ -C₄ alkyl and benzyl; and x is
 1. 26.A method in accordance with claim 23 in which R²⁴, R²⁵, R²⁶ and R²⁷ areeach H; and x is
 1. 27. A method in accordance with claim 23 in whichR²⁴ and R²⁵ are independently selected from the group consisting of H,C₁ -C₈ alkyl, phenyl and benzyl; and x is zero.
 28. A method inaccordance with claim 23 in which R²⁴ and R²⁵ are independently selectedfrom the group consisting of H, C₁ -C₄ alkyl and benzyl; and x is zero.29. A method in accordance with claim 23 in which R²⁴ and R²⁵ are eachH; and x is zero.
 30. A method in accordance with claim 23 in which theR²² and R²³ moieties are each independently selected from the groupconsisting of H, C₁ -C₄ alkyl and benzyl.
 31. A method in accordancewith claim 23 in which the R²² moieties are each H; and the R²³ moietiesare each independently selected from the group consisting of H and C₁-C₄ alkyl.
 32. A method in accordance with claim 23 in which the R²²moieties are each H; and the R²³ moieties are each independentlyselected from the group consisting of H and methyl.
 33. A method inaccordance with claim 23 in which the R²² moieties are each H; and theR²³ moieties are each H.
 34. A method in accordance with claim 23 inwhich R²⁴ and R²⁵ are each H; x is zero; the R²² moieties are each H;and the R²³ moieties are each H.
 35. A method in accordance with claim23 in which R²⁴, R²⁵, R²⁶ and R²⁷ are each H; x is 1; the R²² moietiesare each H; and the R²³ moieties are each H.
 36. A method in accordancewith claim 23 in which R²⁴, R²⁵ and R²⁶ are each H; R²⁷ is OH; x is 1;the R²² moieties are each H; and the R²³ moieties are each H.
 37. Amethod in accordance with claim 23 in which R²⁴, R²⁵ and R²⁶ are each H;R²⁷ is NH₂ ; x is 1; the R²² moieties are each H; and the R²³ moietiesare each H.
 38. A method in accordance with claim 23 in which saidparamagnetic metal cation of a cation of an element having an atomicnumber of 22 to 29 or 58 to
 70. 39. A method in accordance with claim 23in which said paramagnetic metal cation is a cation of an elementselected from the group consisting of chromium, manganese, iron andgadolinium.
 40. A method in accordance with claim 23 in which saidphysiological compatible salt is comprised of said complex incombination with at least one cation selected from the group consistingof sodium and N-methylglucamine.
 41. A method in accordance with claim23 in which said physiologically compatible salt is the combination ofthree equivalents of a physiologically compatible cation with thetrianionic complex of Fe(III) andN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane at a pHof about 6.8 to about 7.4.
 42. A method in accordance with claim 23 inwhich said physiologically compatible salt is the trisodium salt of theFe(III) complex ofN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane.
 43. Amethod in accordance with claim 23 in which said physiologicallycompatible salt is the trimeglumine salt of the Fe(III) complex ofN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane.
 44. Amethod in accordance with claim 23 in which said physiologicallycompatible salt is the trisodium salt of the Fe(III) complex ofN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane.
 45. Amethod in accordance with claim 23 in which said physiologicallycompatible salt is the trimeglumine salt of the Cr(III) complex ofN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane.
 46. Amethod in accordance with claim 23 in which said physiologicallycompatible salt is the tetrasodium salt of the Mn(II) complex ofN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane.
 47. Amethod in accordance with claim 23 in which said physiologicallycompatible salt is the trisodium salt of the Mn(III) complex ofN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane.
 48. Amethod in accordance with claim 23 in which said physiologicallycompatible salt is the trisodium salt of the Gd(III) complex ofN,N',N"-tris(dihydroxyphosphorylmethyl)-1,4,7-triazacyclononane.